CD4+ helper T (Th) cells are critical mediators in autoimmune diseases, such as arthritis, psoriasis and lupus. Upon activation, naove Th cells differentiate into effector cell subsets that are characterized by their distinct gene expression and immunoregulatory function. In the past several years, a number of novel Th subsets have been identified, including Foxp3+ regulatory T cells, pro-inflammatory Th17 cells and T follicular helper (Tfh) cells. These cells not only have been shown to be important positive or negative regulators in autoimmune diseases, but also share reciprocal developmental regulation. TGF2 signaling in activated T cells induces Foxp3 expression, while the combination of TGF2 and IL-6 or IL-21 drives Th17 cell differentiation. On the other hand, we recently reported that IL-6 or IL-21, in the absence of TGF2 signaling, drives Tfh cell differentiation. How STAT3, a common transcription factor downstream of IL-6 and IL-21, and TGF2-activated SMAD transcription factors interplay in T cell fate decision has not been understood. Our data suggest that they may mediate the upregulation of lineage-specific transcription factors, such as Foxp3 in Treg cells, ROR1/3 in Th17 cells and Bcl6 in Tfh cells. Although these cell-specific transcription factors have been shown by others and us to be essential in Th cell lineage specification, the exact mechanisms of their action have not been understood either, nor their cross-inhibitory functions. The proposed work is based on our well-established, multi-disciplinary collaborative team that has made important contributions in the past five years. The central goal of this project is to perform large-scale, genome-wide transcriptome and epigenome analysis of Treg, Th17 and Tfh cells to determine the direct functions of key transcription factors and their likely agonistic and antagonistic interactions. Our central hypothesis is that transcriptional and epigenetic mechanisms induced by key transcription factors underscore the specification and plasticity of novel Th lineage cells. To address this hypothesis, we will perform transcriptome analysis of T cells deficient in TGF2 and STAT3 signaling pathways or Foxp3, ROR1/3 and Bcl6 transcription factors. Moreover, we will perform genome-wide epigenome analysis of T cells deficient in TGF2 and STAT3 signaling pathways or Foxp3, ROR1/3 and Bcl6 transcription factors. Thirdly, we will perform genome-wide chromatin immunoprecipitation-sequencing analysis on the targets of SMAD, STAT3, ROR3/1 and Bcl6. The results of these experiments will be subject to bioinformatic analysis to understand Th lineage specification and plasticity. The project is highly feasible and can be accomplished within the two-year time frame as all the necessary reagents are in hand and methods are well established. The proposed project will provide systemic insights into Th cell lineage commitment and cellular plasticity. The study suits the mission of NIAMS by analyzing the critical cell types that positively or negatively regulate the pathogenesis of skin and rheumatic autoimmune diseases.